tbuckley/cabot-search huggingface dataset:
Abstract P1059: Establishing The Mechanistic Basis Of Dilated Cardiomyopathy Associated With The Skeletal Muscle Actin Mutation R256H
Authors: Ankit Garg, Kory Lavine, Silvia Jansen, Michael Greenberg
Journal: Circulation Research (2022)

Actin, which is expressed in all eukaryotic cells, is an essential component of the cardiac and skeletal muscle sarcomere thin filament. Mutations in actin have been associated with a wide range of diseases, including skeletal myopathies and cardiomyopathies. Humans express six actin isoforms with two that are thought to be highly enriched in sarcomeres and differ by only four amino acids: skeletal muscle actin (ACTA1) and cardiac actin (ACTC1). While ACTC1 is exclusively expressed in the adult heart, ACTA1 is predominantly expressed in skeletal muscle with lower levels detected in cardiac muscle. As such, ACTA1 mutations are well known to cause skeletal myopathy with minimal effects on cardiac function. However, I previously reported a family carrying a novel heterozygous mutation in ACTA1 (ACTA1 R256H) with dilated cardiomyopathy (DCM) and no clinical evidence of skeletal myopathy. The objective of this study is to elucidate the molecular mechanism(s) by which ACTA1 R256H may cause heart failure. I generated ACTA1 R256H/+ human pluripotent stem cells (hPSCs) using Cas9/CRISPR gene editing. Using traction force microscopy, I observed that ACTA1 R256H/+ hPSC-derived cardiomyocytes display reduced contractility. To define relevant mechanisms, I devised a novel approach to purify milligram quantities of recombinant human ACTA1, which has served as a remarkable challenge for the field. Importantly, this purification technique circumvents contamination from endogenous actins and produces functional protein capable of polymerization. Moreover, in vitro motility assays demonstrated that reconstituted human thin filaments containing recombinant ACTA1 are functional and activated in a calcium-regulated manner. Intriguingly, purified ACTA1 R256H appears to have a polymerization defect, suggesting a possible mechanism by which this mutation leads to a defect in contractility. Together, these studies suggest for the first time that a mutation in skeletal muscle actin, ACTA1 R256H, causes cardiomyocyte hypocontractility while also suggesting a potential molecular mechanism. Finally, this study also introduces a new purification method essential for biochemical and biophysical analysis of human ACTA1 mutations.
Abstract P1055: Co-chaperone Bag5 Protects Cardiomyocytes From Oxidative Stress
Authors: Avni Mukker, Puneet Randhawa, Isabela Beatriz de Lima, Manish K Gupta
Journal: Circulation Research (2022)

Introduction: Cardiovascular disease is one of the leading causes of death globally. Cardiac ischemic can alter cellular homeostasis and potentially lead to heart failure. Ischemic cells modulate the expression of molecular chaperones, which assist with the folding/refolding of cellular proteins. Specifically, the heat-shock protein 70 (Hsp70) chaperone, and its co-chaperones in the Bcl-2-associated athanogene (BAG) protein family, help maintain protein homeostasis by protein folding and degradation. This project serves to explore the role of BAG5 in cardiac ischemia. Methods: Expression of BAG5 was determined in left ventricular tissue isolated from cardiomyopathy patients and in ischemic cardiomyocytes. Co-immunoprecipitation (IP) and mass spectrometry analysis were utilized to determine potential interacting partners of BAG5. For the generation of ischemic conditions, neonatal rat cardiomyocytes were incubated in a hypoxia chamber for 12 hours in DMEM media with no glucose, 0% serum, and 0.1% O2. The role of BAG5 in cellular viability, reactive oxygen species (ROS) production, and proteostasis during ischemia was determined through gain-in and knock-down approaches using adenoviruses. In addition, mitochondrial membrane potential and ROS production were determined by the TMRE and MitoSOX red staining, respectively. Mitochondrial function was determined by a Seahorse XF assay. Results: Co-immunoprecipitation and mass spectrometry analysis revealed that BAG5 interacts with Myosin and Hsp70. Expression of BAG5 in cardiomyocytes was significantly decreased during ischemia. Further, overexpression of BAG5 protects cardiomyocytes by inhibiting excessive autophagy and reducing ROS production. Moreover, BAG5 expression improves mitochondrial function during ischemia. Conclusion: Understanding the role of BAG5 within the cell may help establish the protein as a potential therapeutic molecule for the protection of the ischemic heart.
Abstract P3052: Dysregulation Of The Ampk-drp1 Axis Promotes Pathophysiological Remodeling Of Mitochondrial Ultrastructure And Ventricular Electrophysiological Function
Authors: Michael W Rudokas, Marine Cacheux, Xiaohong Wu, Lawrence H Young, Fadi G Akar
Journal: Circulation Research (2022)

AMP-activated protein kinase (AMPK), a master metabolic sensor, plays a central role in the regulation of mitochondrial (mito) function. AMPK activation inhibits mito fission potentially by modulating mito dynamics proteins, including dynamin-related protein 1 (DRP1). The role of the AMPK-DRP1 axis in electrophysiological (EP) function is unknown. Methods: We examined the impact of myocardial AMPK inactivation on mito ultrastructure by electron microscopy, mito dynamics proteins by western blot, and EP function by optical mapping in mice expressing an AMPK kinase dead isoform (AMPK-KD, N=3-7) vs littermate controls (Ctrl, N=3-6). Results: Quantification of mito morphology revealed increased mito area consistent with swelling (0.53 μm 2 vs 0.80 μm 2 , p&lt;0.0001) and decreased mito count (0.68 mito/μm 2 vs 0.44 mito/μm 2 , p=0.13) in AMPK-KD vs Ctrl. Immunoblots revealed decreased DRP1 phosphorylation at S637 (a fission inhibiting site) with no major change in total DRP1, S616 phospho-DRP1, MFN1/2 or OPA1 levels in AMPK-KD. High resolution optical mapping of ex vivo perfused hearts uncovered marked (by 39.5%, p&lt;0.001) conduction slowing ( Figure ) and a moderate increase in AP duration (43.8 ms vs 49.7 ms, p&lt;0.05) reflecting widespread ventricular EP remodeling in AMPK-KD. Of note, discrete lines of functional conduction block were observed in 4/7 AMPK-KD but not Ctrl hearts consistent with pro-arrhythmic remodeling. Conclusions: Myocardial AMPK pathway inactivation causes mito ultrastructural abnormalities and pathological EP remodeling. The AMPK-DRP1 axis may be a novel target for arrhythmia suppression in metabolic disorders with defective AMPK downstream signaling.
Abstract P1001: IGFBP2 Overcomes Cell Contact-mediated Proliferation Inhibition In Human IPSC-derived Cardiomyocytes
Authors: Soah Lee, Paul Heinrich, Daniel Lee, William R Goodyer, Francisco X Galdos, Han Zhu, Tahmina Samad, Sidra Xu, Carissa Lee, Lauren Duan, Sean M Wu
Journal: Circulation Research (2022)

Introduction and Hypothesis: Human induced pluripotent stem cells (hiPSCs) and their derivatives provide a promising cellular source for regenerative cardiac therapies. However, generating large-scale hiPSC-derived cardiomyocytes (hiPSC-CMs) remains a major hurdle, caused, in part, by cell contact-driven proliferation inhibition in dense culture conditions. We hypothesize that differentially-secreted factors in sparsely vs densely-cultured hiPSC-CMs are responsible for the increased proliferation rate in sparsely cultured hiPSC-CMs. Methods: We performed a high-throughput screening (HTS) assay to identify growth factors (GFs) released in the supernatant of sparsely- and densely-cultured hiPSC-CMs. Differential expression of candidate GFs in dense vs sparse culture conditions was confirmed by single cell RNA sequencing (scRNAseq), Western Blot (WB), and Immunocytochemistry (ICC) analyses. The confirmed GF hits were further validated by measuring the expression of the cell cycle marker Ki67 in hiPSC-CMs after GF treatment. The GF treatment effects on cell-cell contact was further explored mechanistically using siRNA-based N-cadherin (CDH2) knockdown (KD) model in vitro. Results: Our HTS assay identified IGFBP2, IGFBP6, PDGF-AA as top candidate GFs in the sparse culture supernatant. Subsequent WB, ICC, scRNAseq analyses confirmed 1.5, 3.5, 2.0-fold higher IGFBP2 expression in the sparse condition compared to the dense condition. Supplementation of recombinant IGFBP2 to densely cultured hiPSC-CMs showed a dosage-dependent increase in Ki67 expression level from 13.0 +/- 1.4% in control to 44.1 +/- 8.4% after 3nM IGFBP2 treatment, a level comparable to that achieved in sparse culture. Mechanistically, IGFBP2 treatment resulted in a shift of CDH2 localization from cell-cell junction to the cytoplasm. Reducing CDH2 expression by CDH2-KD to mimic loss of cell-cell contact promoted hiPSC-CM proliferation. Conclusion: We discovered an unexpected role of IGFBP2 to overcome cell contact-mediated inhibition of hiPSC-CM proliferation which provides a promising new approach to engineer in situ large-scale 3D cardiac tissues for regenerative applications.
Abstract P1079: S100A1-SUMO Interaction Via SUMO Interaction Motif (SIM) Of The S100A1 C-terminus Domain Is Critical For S100A1 Post-translational Protein Stability
Authors: Zegeye Hailu Jebessa, Manuel Glaser, Andrea Schneider, Jemmy Zhao, Ramkumar Seenivasan, Martin Busch, Rebecca Wade, Patrick Most
Journal: Circulation Research (2022)

Background &amp; Objectives: S100A1 protein is a 10 kDa Ca 2+ sensor &amp; abundantly expressed in cardiomyocytes (CMs) of vertebrates. Marked reduction in S100A1 level is hallmark of diseased heart, vice versa, addition of S100A1 by gene transfer rescues diseased heart contractile dysfunction. Since our understanding of the molecular circuits that may contribute to the regulation of S100A1 protein levels are still scarce, we conducted a hybrid computational structural &amp; experimental approach to unveil underlying molecular residues &amp; mechanisms that may control S100A1’s protein stability. Methods &amp; Results: We employed in silico computational prediction &amp; molecular docking tools, respectively, to inform experimental approaches in order to characterize residues within the 94 amino acid (aa) of S100A1. A web server-based GPS-SUMO 2.0 analysis of the human S100A1 sequence unveiled a putative SUMO interacting (SIM) motif (76-VVLVA-80) within the alpha-helical C-terminus of S100A1. Restrained docking with HADDOCK predicted a molecular interaction between SUMO-1 &amp; the SIM-lined groove of the Ca2+-bound (holo) S100A1 homodimer that presents a potential novel type of interaction mode. We then performed an in vitro S100A1-SUMO interaction assay in the presence of 1 mM Ca 2+ or 1 mM EGTA &amp; the assay revealed calcium dependent specific S100A1-SUMO proteins interaction. Overexpression of S100A1 together with SUMO1 increases S100A1 protein abundance in CMs &amp; COS1 cells without changing the mRNA level of S100A1. Overexpression assays in COS1 cells &amp; CMs involving S100A1 truncation mutant lacking SIM motif (S100A1-1-74) or site directed mutagenesis deleted (DSIM) or Alanine replaced SIM of S100A1 showed that S100A1 lacking SIM motif either via truncation &amp; deletion or Alanine substitution led to, respectively, absence of detection or massively reduced overexpression of S100A1 protein without affecting the mRNA overexpression of the mutants. The aforementioned mutants could be rescued at protein level in CMs &amp; COS1 cells by addition of proteasome inhibitor-MG-132. Conclusion: Here we describe a yet unrecognized post-translational molecular checkpoint for S100A1’s protein stability involving a SIM-mediated interaction between S100A1 &amp; SUMO
Rating respiratory disability: a report on behalf of a working group of the European Society for Clinical Respiratory Physiology
Authors: JE Cotes
Journal: European Respiratory Journal (1990)

A rating procedure for respiratory disability has been developed; it entails measuring the symptom-limited maximal oxygen uptake or estimating the maximal uptake from the results of a submaximal exercise test and other relevant variables. The derivation assumes a linear scale of disability between the limits 0% and 100% which are defined. The percentage disability of 157 men with respiratory limitation of exercise has been used to delineate empirical grades of disability. These are of similar form to those used for grading respiratory impairment. More information is needed with a view to validation.
Abstract P3137: Drp1-dependent Mitochondrial Fission Induces Vascular Smooth Muscle Cell Dysfunction Through Kruppel-like Factor 4 Upregulation
Authors: Diem Thi Ngoc Huynh, Yujin Jin, Thuy Le Lam Nguyen, Kyung-Sun Heo
Journal: Circulation Research (2022)

Introduction: Vascular smooth muscle cell (VSMC) phenotypic switching and subsequent VSMC proliferation and migration are major events that are closely associated with progression of cardiovascular diseases. Mitochondria play key roles in regulation of cell function. Recent findings have revealed that mitochondrial fission promotes proliferation and migration of VSMCs. Hypothesis: This study aimed to explore the role of a new drug candidate, ginsenoside Rh1, in Angiotensin II (Ang II)-induced VSMC migration and proliferation and its underlying mechanisms. Methods: Sulforhodamine B and wound-healing assay were used to evaluate effects on cell proliferation and migration. Molecular mechanisms were investigated using western blotting, qRT-PCR analysis, immunofluorescence staining, and luciferase assay. Reactive oxygen species (ROS) production was measured by dihydroethidium and MitoSOX staining. Results: We found that pretreatment with 50 μM Rh1 significantly suppressed Ang II-induced cell proliferation (% of decrease vs. Ang II group: 17.41 ± 2.38%, p &lt; 0.01, n = 5) and migration (% of decrease vs. Ang II group: 198.8 ± 2.67%, p &lt; 0.001, n = 5). Concomitantly, Ang II-increased both protein and mRNA levels of MMP2, MMP9, PCNA, and cyclin D1 were reduced by Rh1 pretreatment. Rh1 incubation also reversed the changes in both protein and mRNA expressions of α-SMA, vimentin, and osteopontin induced by Ang II. Besides, Ang II enhanced protein expression and promoter activity of KLF4, a key regulator of phenotypic switching, whereas pretreatment with Rh1 reversed these effects. Mechanistically, inhibitory effects of Rh1 were found to be associated with inhibition of ERK1/2 signaling pathway and ROS generation. In addition, deactivation of ERK1/2 suppressed phosphorylation of DRP1 at Ser 616. Notably, inhibition of DRP1-dependent mitochondrial fission by Mdivi-1 inhibited KLF4 expression and transcriptional activity induced by Ang II. Conclusions: In conclusion, mitochondrial fission involves Ang II-induced VSMC proliferation and migration through KLF4 upregulation, and that Rh1 inhibits VSMC dysfunction by suppressing ROS-mediated ERK1/2/DRP1/KLF4 signaling pathway.
Abstract P1094: Sexual Dimorphism In Histone Deacetylases Activity Regulates Ventricular Calcium Handling Properties
Authors: Jonathas F Almeida, Kathrin Banach
Journal: Circulation Research (2022)

Introduction: There is a sexual dimorphism in cardiac excitation contraction coupling (ECC) with female ventricular myocytes (F VM ) exhibiting an attenuated basal calcium concentration ([Ca] i ), Ca transient (CaT) amplitude, and a prolonged CaT decay constant (τ). Increased expression levels of the sodium-calcium exchanger (NCX) have been described in F VM however, the role NCX expression plays in sexual dimorphism of physiological and pathophysiological Ca handling properties, and how NCX expression is regulated in a sex specific manner remains to be determined. Here we tested the hypothesis, that a sexual dimorphism in HDAC activity regulates NCX expression and NCXs differential contribution to the CaT adds to the sexual dimorphism in ventricular ECC. Method: [Ca] i was quantified in field stimulated, freshly isolated ventricular myocytes (VMs) from male (M VM ) and female (F VM ) FVB/N mice loaded with the Ca sensitive fluorescent dye Fluo-4/AM. Changes in NCX expression were quantified by qPCR and western blotting. Results: Under basal conditions F VM exhibited an attenuated CaT amplitude (ΔF/F 0 ; M VM :1.8±0.08, F VM :1.4±0.06, p=0.038) and prolonged CaT decay constant (τ: M VM :0.37±0.01ms, F VM :0.46±0.02ms, p&lt;0.01). The caffeine induced Ca transient (CaT caf ) amplitude was comparable in F VM and M VM , but the CaT caf decay constant (τ caf: M VM : 3.34±0.22s ; F VM :4.81±0.36s) was longer in F VM although increased NCX expression was determined on the protein and mRNA level. Consistent with increased NCX expression, F VM showed an attenuated increase in basal [Ca] i (F/F 0 ; M= 60%, F=44%, p=0.0033) and τ (M=0.37±0.01s, F=0.46±0.02s, p&lt;0.0001) when SERCA activity was attenuated (CPA: 10μM). Also during conditions favoring NCX reverse mode ([Na] o =93, 46 mM) F VM exhibited a larger increase in CaT amplitude (ΔF/F 0 ; M=81%, F=133%, p&lt;0.01) and an increased propensity for spontaneous Ca release events. Inhibition of HDAC class II (LMK: 5mg/kg/day) did not change Ca handling properties in F VM but slowed τ (M LMK : 0.44±0.03ms) and τ caf in M VM (M LMK =5.1±0.33s) making them comparable to F VM . Conclusion: The sexual dimorphism in ventricular ECC is mediated by attenuated HDAC activity in F VM s and facilitates NCX Ca overload.
Histiocytosis X characterized by marked elevation of serum alkaline phosphatase and rapid destructive changes in the lung parenchyma
Authors: PB Hansen
Journal: European Respiratory Journal (1989)

A 23 yr old man was admitted suspected of having a liver disease because of marked elevation of the serum alkaline phosphatase. A biopsy of the liver was without cholestasis. A skin biopsy of a papule was compatible with eosinophilic granuloma. Over a few months the patient developed severe restrictive lung disease. The elevation of the serum alkaline phosphatase was probably due to diffuse bone affection.
Abstract P2079: Rbl2 Regulates Cardiac Sensitivity To AnthracyclineChemotherapy
Authors: Jingrui Chen, Peng Xia, Yadav Sapkota, Erika N Scott, Yuening A Liu, Melissa Hudson, Shahrad R Rassekh, Bruce C Carleton, Colin Ross, Eric Chow, Zhaokang Cheng
Journal: Circulation Research (2022)

Anthracycline chemotherapies cause heart failure in a subset of cancer patients. Emerging evidence suggests that genetic factors might contribute to the interindividual variations in cardiac sensitivity to anthracyclines. We previously reported that the anthracycline doxorubicin (DOX) induces cardiotoxicity through activation of cyclin-dependent kinase 2 (CDK2). The aim of this study was to determine whether retinoblastoma-like 2 (RBL2/p130), an emerging CDK2 inhibitor, regulates CDK2 activity and anthracycline sensitivity in the heart. Here, we showed that loss of endogenous Rbl2 increased basal cardiac CDK2 activity. Mice lacking Rbl2 were more sensitive to DOX-induced cardiotoxicity, as evidenced by rapid deterioration of heart function and loss of heart mass. Disruption of Rbl2 exacerbated DOX-induced mitochondrial damage and cardiomyocyte apoptosis. Mechanistically, Rbl2 deficiency enhanced CDK2-dependent activation of forkhead box O1 (FOXO1), leading to upregulation of the pro-apoptotic protein Bim. Inhibition of CDK2 desensitized Rbl2-depleted cardiomyocytes to DOX. In wild-type cardiomyocytes, DOX exposure induced Rbl2 expression in a FOXO1-dependent manner. Importantly, human RBL2 gene single nucleotide polymorphism (rs17800727) was also associated with anthracycline cardiotoxicity in childhood cancer survivors. In conclusion, loss of Rbl2 provokes cardiac CDK2 activation, resulting in increased sensitivity to DOX-induced cardiotoxicity. Rbl2 is an endogenous CDK2 inhibitor in the heart and represses FOXO1-mediated pro-apoptotic gene expression. Our findings suggest that RBL2 could be used as a biomarker to predict the risk of cardiotoxicity in individual patient prior to initiation of anthracycline-based chemotherapy.
Abstract P2066: Age-related Changes In Posttranscriptional Regulation Of Macrophage VEGF As A Mechanism Of Impaired Inflammatory Arteriogenesis
Authors: Chris S Mantsounga, Sheila Sharma, Cadence Lee, Jade Neverson, Rachel Carley, Gaurav Choudhary, Alan R Morrison
Journal: Circulation Research (2022)

Aging and age-related diseases like peripheral arterial disease are associated with impaired inflammatoryarteriogenesis in response to injury. During ischemia, macrophage recruitment and macrophage pro-angiogenic VEGF-A isoform expression contribute to post developmental arteriogenesis. Preliminary datafrom bone marrow-derived macrophages (BMDMs) demonstrated decreased VEGF-A level by both mRNAand protein in aged (52-week-old) and advanced aged (104-week-old) mice when compared to young (12-week-old). Moreover, VEGF-A165a which is VEGF-A proangiogenic splice variant was also reduced inmacrophages from aged and advanced aged mice. The anti-angiogenic VEGF-A165b variant wassignificantly increased in advanced aged mice. We sought to determine the mechanisms which cancontribute to the reduction of pro-angiogenic VEGF-A and consequently reduced inflammatory-mediatedarteriogenesis response in the context of aging. In a hindlimb ischemia model of angio/arteriogenesis, wefound decreased blood flow recovery in aged mice. Moreover, small arterial angiography by microCTconfirmed decreased arteriogenesis. Loss of functional arteriogenesis was associated with decreased muscletissue VEGF-A and VEGF-A165a levels despite adequate macrophage recruitment to the muscle tissue.Moreover, VEGF-A165b was increased in ischemic tissue from advanced aged mice. The mechanism ofreduced macrophage pro-angiogenic VEGF-A165a expression involved decreased mRNA stability withdecreased association of VEGF-A165a mRNA with the RNA-stabilizing protein HuR. In fact, aged BMDMsdemonstrated a loss of HuR binding to VEGF-A165a mRNA with decreased VEGF-A165a mRNA half-life,and consequently decreased inflammatory angio/arteriogenesis.